EFFECT OF TREATMENT WITH BENZIMIDAZOLE ANTHELMINTICS ON THE BALAGANGADHARREDDY REDDYJARUGU. (Under the Direction of Ray M.

Transcription

1 EFFECT OF TREATMENT WITH BENZIMIDAZOLE ANTHELMINTICS ON THE VIABILITY OF BRUGIA MALAYI: IN VITRO AND IN VIVO STUDIES by BALAGANGADHARREDDY REDDYJARUGU (Under the Direction of Ray M. Kaplan) ABSTRACT The concentration and time point at which lethal effects in Brugia malayi adult female worms are observed following treatment with different concentrations of thiabendazole were studied in vitro by measuring worm motility, microfilariae release and MTT assay. Results showed thatworm motility and microfilariae release assays are the best indicators of worm viability, still leaving worms usable for gene expression studies; and motility assay determines worm viability quicker than mf release assay for gene expression studies. The concentration and time points, at which B. malayi worms show varying level of susceptibility to albendazole (ABZ), were determined in vivo. Moderately active worms with increased drug susceptibility at 21 days posttreatment (DPT) and highly active worms with greater tolerance at 28 DPT were selected. RNA was extracted from these worms for microarray analysis to gain basic knowledge of gene expression in B. malayi that survived the treatment compared to worms susceptible to ABZ. INDEX WORDS: Lymphatic filariasis, Brugia malayi, benzimidazole, worm viability, in vitro and in vivo

2 EFFECT OF TREATMENT WITH BENZIMIDAZOLE ANTHELMINTICS ON THE VIABILITY OF BRUGIA MALAYI: IN VITRO AND IN VIVO STUDIES by BALAGANGADHARREDDY REDDYJARUGU B.V.Sc. & A.H., ANGRA University, India, A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2008

3 2008 BALAGANGADHARREDDY REDDYJARUGU All Rights Reserved

4 EFFECT OF TREATMENT WITH BENZIMIDAZOLE ANTHELMINTICS ON THE VIABILITY OF BRUGIA MALAYI: IN VITRO AND IN VIVO STUDIES by BALAGANGADHARREDDY REDDYJARUGU Major Professor: Committee: Ray M. Kaplan Julie M. Moore David S. Peterson Michael Dzimianski Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2008

5 DEDICATION To my wife, Prachi, our son, Saket and our parents for their love and support. iv

6 ACKNOWLEDGEMENTS I would like to thank my major professor Dr. Ray M. Kaplan for his able guidance and endless patience. He has been a constant source of inspiration and challenge for me all through my graduate study. I would like to thank my committee members Drs. Julie M. Moore, David S. Peterson and Michael Dzimianski for their invaluable suggestions. I would also like to thank my lab colleagues Drs. Dzimianski, Supakorndej, Moorehead, Mr. Bob Storey and Ms. Sue Howell for their much-needed technical help in the laboratory during my research. My time during graduate study would have been very tedious and monotonous without the friendship of Dhanu, Srujana and Krishna. Finally, I would like to thank my family for providing encouragement and moral support during the completion of this work. v

7 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS...v LIST OF TABLES... vii LIST OF FIGURES... viii CHAPTER 1 LITERATURE REVIEW...1 References EFFECT OF TREATMENT WITH BENZIMADAZOLE ANTHELMINTICS ON THE VIABILITY OF BRUGIA MALAYI: IN VITRO...34 References EFFECT OF TREATMENT WITH ALBENDAZOLE ON THE VIABILITY OF BRUGIA MALAYI: IN VIVO...53 References CONCLUSIONS...93 vi

8 LIST OF TABLES Page Table 3.1: Pilot study results: Summary of the worms used in transplantation procedure and motility score of recovered worms...64 Table 3.2: Pilot study results: Worms isolated from the treated used...66 Table 3.3: Summary of the worms used in transplantation procedure and motility score of recovered worms...67 Table 3.4: Worms isolated from the control and treated gerbils...68 Table 3.5: Total numbers of worms isolated from the control and treated gerbils based on their motility scores for RNA extraction...68 Table 3.6: Total RNA isolated from the selected worms...69 vii

9 LIST OF FIGURES Page Figure 2.1 Motility assay:...42 Figure 2.2Microfilariae release assay...43 Figure 2.3 MTT assay:...44 viii

10 CHAPTER I LITERATURE REVIEW 1.1 Parasite Biology and Disease Lymphatic filariasis (LF) is a disease caused by vector borne, filarioid nematode parasites Wuchereria bancrofti, Brugia malayi and Brugia timori (Mark, 1986). The long and thin adult worms (macrofilariae) reside in the lymphatic system. Female worms measure mm in length and µm in width, and male worms measure 13 to 23 mm in length by 70 to 80 µm in width. Fertilized females produce millions of larval forms, called microfilariae (mf), measuring 177 to 230 µm in length and 5 to 7 µm in width, which are covered by an external cuticular sheath and have nocturnal periodicity. The mf migrate from the lymph to the blood stream, reaching peripheral blood vessels where they are ingested by female mosquitoes during a blood meal. Over an approximately 2 week period, the mf molt twice to become 2 nd and 3 rd stage larvae (called the 'L3'). The L3 is the 'infective stage' which are deposited onto the skin of an another host during a subsequent blood meal. The L3 then invades the subcutaneous tissues and migrates to the lymphatics where it matures to the adult stage which live there for seven or more years. These adult worms may find other adults of the opposite sex, and mate resulting in the production of mf, thus continuing the life cycle. A number of mosquito genera and species are able to transmit filariasis, but the two main forms of the disease, bancroftian and brugian filariasis, have distinct primary vectors. The most prominent vector of bancroftian filariasis, caused by W. bancrofti and responsible for most 1

11 (90%) cases of human filariasis, is Culex quinquefasciatus, a mosquito that thrives in urban areas and villages of tropical and subtropical regions (Mak, 1986). Brugian filariasis is caused by B. malayi and its main vectors belong to the genus Mansonia (Evans et al., 1993). B. malayi is limited to Southeast and Eastern Asia (Melrose, 2002) and accounts for 10% of filariasis worldwide. Since, the vectors of B. malayi require freshwater plants for their larvae, they are confined to rural regions (Evans et al., 1993). Traditionally, filarial-infected people are grouped into one of the following categories at a given time: (1) endemic normal- no mf present in the bloodstream and no clinical signs; (2) asymptomatic microfilaraemia-mf found in the blood, but no clinical signs; and (3) chronic filarial disease- mostly amicrofilaraemic, with or without acute attacks (Dasgupta, 1984; Partono, 1987; Ottesen, 1992; Evans et al., 1993; Ottesen, 1994). But people affected with any of these conditions may also suffer from acute filarial disease (Melrose, 2002). Endemic normal refers to people who, despite constant exposure to filariasis and circulating filarial antibodies, are amicrofilaraemic and have no clinical evidence of disease (Ottesen, 1989). But, these individuals may have cryptic infection due to the presence of circulating filarial antigen (Weil et al., 1996). Asymptomatic microfilaraemia is the most commonly seen form of filariasis and despite initial absence of obvious clinical disease, damage to lymphatics and organs may occur in people suffering from it (Freedman et al., 1995a; Freedman et al., 1995b; Senarath et al., 1995). Chronic disease is often manifested as lymphedema (with swollen limbs) progressing to elephantiasis in chronic brugian lymphatic filariasis and hydrocele in chronic bancroftian filariasis (Evans et al., 1993; Witte et al., 1993). Uncommonly, chyluria and Tropical Pulmonary Eosinophilia (also known as 'occult filariasis ) may be seen in chronic filariasis (Evans et al., 1993). Tropical Pulmonary Eosinophilia 2

12 symptoms may include cough dyspnea and may be similar to asthma symptoms (Pinkston et al., 1987). Acute filarial disease usually begins in late childhood or early adulthood and may be caused by dermatolymphangioadenitis (with the development of a plaque-like skin lesion and inflammation of lymphatics) or filarial lymphangitis (due to immune reactions to dead or dying adult worms killed by host immune response or chemotherapy) (Dreyer et al., 1996a; Dreyer et al., 1996b). This type of filariasis is systemically manifested as fever, headache, chills, nausea, vomiting or loss of appetite (Nanduri and Kazura, 1989; Evans et al., 1993). 1.2 The social and economic impact of lymphatic filariasis Filarioid nematodes are among the most important parasites affecting both animals and human health worldwide. The World Health Organization (WHO) estimates that 120 million people are currently infected, with 1.3 billion people at risk of infection in 83 countries throughout the tropics and sub-tropics of Asia, Africa, the Western Pacific, and parts of the Caribbean and South America. The e estimated Disability Adjusted Life Years (DALY) burden due to LF is 5.55 million (Molyneux et al., 2003). WHO has considered LF as the second leading cause of permanent and long-term disability worldwide (WHO, 1997c, b). Lymphatic filariasis is an endemic disease and a major cause of acute and chronic morbidity and incapacitation with devastating public health and socio-economic consequences, associated with conditions of poverty. In 2003, the World Bank classified eighty percent of LF endemic countries as low or lower-middle income countries ( Rural to urban migration and urbanization, occurring increasingly in low income countries, facilitate the spread of LF. This is mostly due to inadequate waste disposal and sanitation facilities, which increase the number of breeding sites for the mosquito vectors (Hunter, 1992; Dhanda et al., 1996). It has been 3

13 calculated that LF causes billions of dollars in lost productivity in India (Ramaiah et al., 2000a) and Africa (Haddix and Kestler, 2000). Reduced productivity from LF disability has been documented in rubber tappers in Malaysia (Kessel, 1957) and agricultural workers in Guyana (Gigliolo, 1960). In endemic rural regions of India, three-fourths of chronic LF patients spend 2% of the average yearly wage on treatment for their conditions and approximately 45% of the total expenditure on medicines (Rao et al., 1982; Ramaiah et al., 2000b; Babu et al., 2002). Stigma and discrimination are byproducts of LF that lead to reduced prospects of both marriage and a normal sexual life (Kessel, 1957). Thus, research on filariasis will have tremendous social and economic benefits. 1.3 Chemotherapy and control programs Two decades ago three barriers to LF elimination existed: lack of- a) tools necessary to interrupt transmission and halt disease progression; b) understanding of disease dynamics and pathogenesis; and c) public awareness of LF s impact and public commitment to overcome it (Ottesen, 2000). In response to this global health crisis, a public-private partnership was developed for the control of LF (WHO, 1997a). This led to the establishment of the Global Program to Eliminate Lymphatic Filariasis (GPELF), which is largely based upon the regular administration of community-wide mass chemotherapy of the entire at-risk population with the anthelmintic albendazole (ABZ) in combination with ivermectin (IVM) or diethylcarbamazine citrate (DEC) (WHO, 1997a, 1999). Three events became critical building blocks of this program s foundation. First, in 1993, the International Task Force for Disease Eradication reviewed approximately 100 medical conditions (mostly infectious diseases) and six of these diseases, including LF, were categorized as eradicable or potentially eradicable with the tools then available (CDC, 1993). This created 4

14 awareness and interest in LF elimination and enhanced its plausibility among the scientific and public health communities. Second, in 1997 the World Health Assembly (WHA) called for the countries to strengthen activities toward eliminating LF as a public health problem and mobilize support for global and national elimination activities. The importance of this resolution was key in its collective recognition of both the achievements to date and the potential benefits to be gained from local and global LF elimination programs. Third, following on the heels of the WHA resolution (50.29), which called for the formation of a public health program focusing on elimination of LF, three additional events catalyzed an exponential increase in the number of persons treated for LF. These events were: 1) donation of ABZ globally by GlaxoSmithKline; 2) donation of Mectizan (IVM) (Merck and Co. Inc.) for countries where LF and onchocerciasis (river blindness) are co-endemic, for as long as needed and; 3) a US $20 million grant from the Bill & Melinda Gates Foundation for LF elimination (Ottesen et al., 1997; Molyneux et al., 2000). This led to the establishment in early 2000 of the GPELF, which had two major goals: to interrupt transmission of the parasite and to alleviate and prevent both suffering and disability caused by the disease. To interrupt transmission of infection, the entire at risk population (WHO, 1997a, 1999) must be treated for a period long enough to ensure that levels of mf in the blood remain below those necessary to sustain transmission. Three drugs shown to be safe and effective in killing the mf are DEC,IVM and ABZ (Addiss et al., 1997; Eberhard et al., 1997; Ismail et al., 1998) and their yearly, single-dose regimens include combinations of- ABZ (400mg) and IVM ( mg/kg/body wt) or ABZ (400mg) and DEC (6mg/kg/body wt) ( 5

15 The principal strategy to alleviate suffering and decrease the disability caused by LF is focused on decreasing secondary bacterial and fungal infections in LF. Secondary infections may worsen the lymphedema and elephantiasis in genitals and limbs-with lymphatic function compromised by filarial infection. Hygiene in treatment of affected areas and creating hope and understanding among the patients, caregivers and their communities are also used (Mathieu et al., 2004). 1.4 Benzimidazoles Anthelmintics are drugs that expel helminths which include cestodes, nematodes and trematodes from the body, by either killing or paralyzing the organisms. Three major classes of anthelmintics include: benzimidazoles (BZ), tetrahydropyrimidines, and macrocyclic lactones. Benzimidazoles are widely used for the control of nematode infections in animals and humans, and include the drugs ABZ, mebendazole (MBZ) and thiabendazole. Benzimidazoles are commonly used because of their high therapeutic index, low price and the absence of toxic residues in food animals (Brown, 1961; McKellar and Scott, 1990). The first BZ to be developed and licensed for animals use in sheep in United States of America was thiabendazole in 1961(Brown, 1961). These drugs are believed to act by binding to the cytoskeletal heterodimeric protein- tubulin, and blocking the formation of the microtubule matrix-an essential component of all eukaryotic cells (Friedman, 1979; Lacey, 1988b). Microtubules play an important role in several important cell functions such as cell division, structure, motility and intracellular substrate transport. The tubulin dimer is comprised of two closely related soluble proteins, α and β-tubulins (Lacey, 1988b). When α-/ β-tubulins with bound BZ are incorporated into the growing end of the microtubule, further heterodimers are prevented from being associated. Since dissociation continues at the opposite end, the microtubules become 6

16 destabilized and finally degraded (Lacey, 1988b, 1990; Lacey and Gill, 1994a). β-tubulins consist of a highly conserved polypeptide framework, with regions of distinctly clustered amino acid substitutions, particularly at the carboxyl terminus (Lopata and Cleveland, 1987; Sullivan, 1988). β-tubulins are grouped into six isotype classes in birds and mammals based on differences in carboxy-terminal sequences and each isotype represents a structurally unique protein sequence encoded by a distinct member of the gene family (Sullivan, 1988). Several nematodes, including C. elegans (Driscoll et al., 1989), B. pahangi (Guenette et al., 1991), H. contortus (Geary et al., 1992a) and Trichostrongylus colubriformis (Grant and Mascord, 1996), possess more than one β-tubulin isotype, but except for some differences in carboxy-termini, β-tubulin sequences are highly conserved in them (Driscoll et al., 1989; Guenette et al., 1991). Despite close sequence homology, nematodes and mammals are differentially sensitive to tubulin inhibitors like BZs, which bind differentially to nematode tubulin compared to mammalian tubulin (Lacey, 1988b; Geary et al., 1992a). This may at least partially account for the selective clinical utility of BZs in controlling nematode infections (Geary et al., 1992b). Of all the BZ drugs, ABZ is the one most commonly used in LF elimination programs. The filariasis elimination strategy requires a community-wide treatment in endemic areas of all at risk' for infection and not just those with documented filariasis (Ottesen et al., 1997). Albendazole, which is co-administered with either DEC or IVM, has micro- and macrofilaricidal effect (Ottesen et al., 1999) and is also effective in treating intestinal helminth infections (Albonico et al., 1994). Therefore, the inclusion of ABZ into a two-drug treatment regimen for the control or elimination of LF may result in a public health impact far greater than LF elimination alone (Ottesen et al., 1999). 7

17 1.5 Benzimidazole resistance Drug resistance is defined as when a greater frequency of individuals in a parasite population, usually affected by a dose or concentration of compound, is no longer affected or a greater concentration of drug is required to reach a certain level of efficacy (Wolstenholme et al., 2004). This is caused by a change in gene frequency of that population resulting from drug selection and alleles for resistance being inherited by the next generation (Prichard et al., 1980). Molecular mechanisms of BZ resistance are very well studied compared to other major anthelmintic classes (Gilleard, 2006). It has been discovered that certain β-tubulin sequence polymorphisms that result in amino acid sequence changes and the loss of high affinity receptor binding sites cause resistance against the BZs (Lacey and Gill, 1994b). A single nucleotide polymorphism (SNP) at codon 167 or 200 of the β-tubulin isotype-1 results in the change to amino acid tyrosine (TAC) instead of phenylalanine (TTC) in resistant isolates as compared to the susceptible worms (Kwa et al., 1993b, 1994; Prichard, 2001; Silvestre and Cabaret, 2002), though substitution at position 200 is more common in parasitic nematodes (Pape et al., 1999; Prichard, 2001; Silvestre and Cabaret, 2002). This subtle mutation was repeatedly found to correlate with drug resistance in several nematodes including H. contortus (Kwa et al., 1993b, 1994; Kwa et al., 1995), Caenorhabditis elegans (Driscoll et al., 1989; Kwa et al., 1995), Cooperia oncophora (Njue and Prichard, 2003) and some equine small strongyles (Pape et al., 2003; Drogemuller et al., 2004b). However, there is increasing evidence that the situation is more complex and other loci may be involved (Lejambre et al., 1979; Kwa et al., 1993a; Kwa et al., 1993b; Roos et al., 1995; Anderson et al., 1998; Sangster et al., 1998; Prichard, 2001). SNPs, also resulting in phenylalanine to tyrosine exchange at codon 167 of the β-tubulin isotypes 1 and 2 in H. contortus (Prichard, 2001) and T. circumcincta (Silvestre et al., 2001), or only isotype 1 8

18 in equine cyathostomins (Drogemuller et al., 2004a), have been found to be closely linked with BZ-resistance. Furthermore, β-tubulin isotype 2 deletion has also been associated with high levels of BZ-resistance (Kwa et al., 1993a). Several reports show an evidence of BZ resistance in human parasitic nematodes too. Evidence of reduced efficacy of BZ anthelmintics against geohelminths has been reported, but has yet to be confirmed by biological or molecular tests (Albonico et al., 2004). In the human filarial nematode, W. bancrofti, the Phe200Tyr SNP has been found in β-tubulin and its prevalence increased dramatically in microfilariae obtained from patients in Burkina Faso, after two yearly doses of combination treatment with ABZ and IVM (Schwab et al., 2005). There is a large body of experimental data demonstrating that the β-tubulin isotype-1gene is strongly associated with BZ resistance in parasitic nematodes (Roos, 1990; Roos et al., 1990; Kwa et al., 1993a; Kwa et al., 1993b; Roos et al., 1995; Elard and Humbert, 1999). However, it is believed that BZ resistance is more complex than this, and is likely polygenic (involving more than one gene). Since worms gain higher levels of resistance with increased drug selection pressure, only a single gene point mutation could not be solely responsible for this phenotype. Benzimidazole resistance is semi-dominant and autosomal in character in H. contortus, and has been suggested to be inherited by more than one gene (Lejambre et al., 1979; Herlich et al., 1981; Anderson et al., 1998). Apart from such target gene (β-tubulin) changes, it has been shown that inhibitors of P-glycoprotein can increase the sensitivity of trichostrongylid nematodes to benzimidazoles suggesting that drug metabolism and drug efflux mechanisms could also play a role in BZ resistance (Gilleard, 2006). 9

19 Why should we be concerned about BZ resistance developing in human filarial nematodes? Benzimidazole anthelmintics have been used extensively in veterinary medicine over the past few decades, but unfortunately resistance to these drugs has made them useless against most of the important nematode parasites of livestock (Kaplan, 2004). Mass drug administration programs against human helminth parasites as seen in GPELF, APOC (African Program for Onchocerciasis Control) and other global programs can put strong selection pressure on those parasites to develop resistance (Prichard, 2007). Several reports suggest that BZ drugs have reduced efficacy against human geohelminthic parasites (Clercq et al., 1997; Reynoldson et al., 1998; Albonico et al., 2003; Albonico et al., 2004) and in W. bancrofti (Schwab et al., 2005), but enough data looking at anthelmintic resistance (AR) in human nematode parasites to understand the extent of existence of drug resistance, if any, is not available. The extent of refugia (proportion of the parasitic population that is not exposed to a particular drug, thereby escaping selection for resistance) at the time of anthelmintic treatment also has a very important role in the selection of AR. In geohelminths, a high proportion of the total population at the time of treatment is on soil as eggs or free-living larval stages, leaving relatively high levels of refugia. Trichostrongylid nematodes of small ruminants and human filarial worms such as W. bancrofti have low levels of refugia at the time of treatment, believed to associate with the development of AR (Swan et al., 1994; van Wyk, 2001; Prichard, 2005). Thus, lower levels of refugia likely cause higher selection pressure for drug resistance in human filarial nematodes as compared to soil transmitted nematodes of humans or animals (Prichard, 2007). 10

20 BZ resistance in parasitic nematodes is recessive (Prichard, 2001) and if non-random mating is seen in these infections, the resistance will develop more rapidly as compared to random mating. Non-random mating(only with their own species), as seen in human filarial nematodes such as W. bancrofti and O. volvulus, increases the proportion of homozygous population, which in turn has been shown to have an increased rate of development of resistance as compared to the heterozygous population (Prichard, 2007). Human filarial nematodes have very long life spans and generation intervals greater than livestock nematodes, which tends to slow the selection for AR. However, all anthelmintics used in mass chemotherapy are slightly/moderately macrofilaricidal (Eberhard et al., 1997; Ismail et al., 1998; Michael et al., 2004) and cause prolonged suppression of reproduction in the surviving adult worms. The susceptible worms are either removed or sterilized and surviving adult worms which can resume reproduction despite repeated treatments produce progeny for a long timeproviding a very strong selection for resistance (Prichard, 2007). A combination of drugs-abz, DEC and IVM used in mass chemotherapy should delay the development of resistance, if the resistance is entirely associated with changes in the drug receptor and the component drugs target different receptors. However, this is not always the case. Macrocyclic lactones such as IVM are potent substrates for P-glycoproteins and ATPbinding cassette (ABC) transporters. ABC transporters, which appear to be involved in IVM resistance (Prichard and Roulet, 2007) are also good receptors for ABZ, suggesting that a combination therapy (ABZ+IVM) could possibly result in enhanced selection of ABC transporters involved in resistance for both ABZ and IVM (Prichard, 2007). Similarly, in human filarial nematodes (O. volvulus and H. contortus) IVM selects β-tubulin (Eng and Prichard, 2005; Eng et al., 2006), which is known to be an excellent substrate for ABZ. A significantly higher 11

21 allele frequency of the resistance-associated 200 tyrosine genotype has been seen in worms collected from patients treated with ABZ in combination with IVM (Schwab et al., 2005). Thus, selection by two anthelmintics can potentially involve the same gene and it can be hypothesized that a combination chemotherapy with ABZ and IVM might actually increase AR selection rather than delay it (Prichard, 2007). Frequent treatment has been portrayed as being important for the selection of AR, but rapid development of AR has been reported in parasites of sheep even in cases in which only two treatments per year were used (Besier and Love, 2003). This took place in a situation where low levels of refugia were present, the condition that prevails in LF when mass drug administration is applied. Furthermore, ABZ exerts a prolonged and continuous effect over several months and could be expected to exert a very high level of selection pressure for resistance (Prichard, 2007). The spread of ABZ resistance is also strongly dependent on treatment coverage. Higher levels of therapeutic coverage would lead to faster mf reductions, but also to quicker spread of ABZ resistance. In small ruminants, where AR has developed rapidly, the practice has been to treat the whole herd. The LF control programs aim to cover 65-85% of the eligible population in a community, which is conducive to the selection for resistance (Prichard, 2005). The model proposed by Schwab et al indicates that increasing the treatment coverage by 10% would lead to an almost 4-fold increase in the frequency of ABZ-resistant mf population after the stopping of 10-yearly treatments. This is because the untreated hosts act as a refugia of susceptible parasites (Schwab et al., 2006). 1.6 Candidate gene approach A major goal in current research is to identify specific genetic changes responsible for and/or associated with AR, which may serve as genetic markers of resistance. These markers 12

22 would allow the resistance to be monitored easily and be identified at an early stage, before genetic changes in the parasite reach a point of significant treatment failure. The major approach for identifying AR-conferring genes is to study candidate genes in susceptible and drug-resistant parasite isolates (Gilleard and Beech, 2007). Candidate genes have a known biological action involved with the development or physiology of a trait. Candidate gene study is based on understanding the drug effects and the physiological response to it. The study involves hypothesizing as to which genes might be involved in the resistance and then conducting experiments to test the hypothesis. Examples of such studies include investigating genetic and biochemical differences between susceptible and resistant gastrointestinal parasites of ruminants and horses and identifying associations of the resistance phenotype with polymorphism in candidate genes (Gilleard, 2006; Gilleard and Beech, 2007) Candidate gene strategy applied to BZ resistance Candidate gene approach in parasitic nematodes has elucidated the role of β-tubulin encoding genes which confer resistance to the BZ class of drugs (Gilleard, 2006; Gilleard and Beech, 2007). β-tubulin is an important target of BZs in a wide range of organisms like nematodes and fungi and mutations in this locus, including F200Y substitution, have been known to provide high levels of resistance to these organisms (Sheir-Neiss et al., 1978; Driscoll et al., 1989; Roos, 1990; Roos et al., 1995; Elard and Humbert, 1999). The candidate gene approach has identified several mutations in this gene and has been highly successful (Gilleard, 2006) Limitations of candidate gene association studies and need of expression-based genomic technologies 13

23 The candidate gene or one gene at a time approach was successful with BZ resistance since the hypothesis chosen was robust. But the same approach has not yet identified a locus that is clearly a major single determinant of IVM resistance in parasitic nematodes (Gilleard, 2006). One reason for this could be that the mechanism of IVM resistance is complex and multigenic, as seen in C. elegans, where a high level of resistance is dependent upon the co-occurrence of mutations in many genes. Other reasons could be that only one or two loci are important, but they have not yet been considered as candidate genes, demonstrating the limitation of this onegene at a time strategy. The candidate gene approach established that mutations in the β-tubulin gene at amino acid positions 167 and 200 correlate with BZ-resistance phenotype. BZ resistance may involve more than one gene (polygenic) (Lejambre et al., 1979; Herlich et al., 1981; Anderson et al., 1998; Gilleard, 2006) and candidate gene strategy is not suitable for polygenic traits. This strategy does not identify novel genes which are responsible for resistance, but are not predicted by the current hypotheses (Gilleard, 2006). Furthermore, if wrong hypotheses or candidate genes are selected, the data generated do not help in understanding the genetics of resistance. Consequently, detailed genetic and population genetic studies are essential to support ongoing work with candidate genes and the future application of genome- wide approaches. Genomic and genetic approaches (gene microarrays, proteomics etc) not only identify the resistance genes, but also assess their relative importance to the resistance phenotype as seen in insecticide resistance (Gilleard, 2006). These approaches require development of good genetic tools and resources, including fully sequenced genomes of parasitic nematodes requiring detailed evaluation of their AR (Gilleard and Beech, 2007). 14

24 1.7 Brugia malayi as a model for parasitic nematodes for gene expression Wuchereria bancrofti is the main species responsible for 90% human lymphatic filariasis (Mak, 1986) whereas B. malayi accounts for 10% of infections worldwide (Evans et al., 1993; Melrose, 2002). Despite its importance, the biology of W. bancrofti is difficult to study due to a lack of an animal model, difficulty in obtaining worm material (Unnasch, 1994) and experimental and ethical limitations of studies of human patient populations. Therefore, B. malayi is generally used as a surrogate in LF research (Unnasch, 1994). Brugia malayi, like other filarial nematodes, develops through five larval stages into an adult male or female, entirely within two host species- a mosquito vector (Culex, Aedes, and Anopheles) and a human host. B. malayi was chosen for sequencing of its entire genome because it can be easily maintained in small laboratory animals, and therefore can be easily manipulated in order to conduct biological experiments and is extremely well characterized biologically (Ghedin et al., 2004a). Several animal models have been used to study Brugia species including dogs, cats, ferrets, rats, mice and gerbils (Schacher and Sahyoun, 1967; Ah and Thompson, 1973b; Ash, 1973; Crandall et al., 1982; Lawrence, 1996; Bell et al., 1999). Each of these models has its own value, and has greatly contributed to understanding the biology of this parasite and the disease it produces. However, Mongolian gerbil (Meriones unguiculatus), models are preferred over others due to maintenance of all parasite life cycle stages, ease of availability, low maintenance costs, and less societal attention to animal experimentation. Hence, gerbil, because of its many characteristics, is a widely used rodent model for Brugia species to study lymphatic filariasis (Ah and Thompson, 1973a; McCall et al., 1973a; Chirgwin et al., 2005). As compared to other animal models, the gerbil model is cheaper, quicker and more efficient in intraperitoneal worm implantation procedures used to study the effects of 15

25 antifilarial drugs (McCall et al., 1973a). In addition, a high percentage (90-95%) of implanted worms can be recovered from this model, reducing the number of gerbils required for the study Brugia malayi draft genome A large expressed sequence tag (EST) effort (over 26,000 ESTs of the significant Brugia life cycle stages have been sequenced) was followed by complete sequencing of the genome of B. malayi (Williams et al., 2000; Ghedin et al., 2004b). The WHO-sponsored Filarial Genome Project (FGP), which was organized in 1994, completed the draft genomic sequence of B. malayi in September Like most other filarial nematodes, B. malayi has three genomes: nuclear, mitochondrial and that of an endosymbiont bacteria, Wolbachia. The nuclear genome of B. malayi is organized into five pairs of chromosomes (4 pairs of autosomes and one pair of sex chromosomes) (Sakaguchi et al., 1983). Using a whole-genome shotgun method, the B. malayi nuclear genome was sequenced at nine fold (9x coverage) redundancy (Ghedin et al., 2004b). The genome has been estimated to be 80 to 100 megabases (Mb) (McReynolds et al., 1986; Sim et al., 1987) and was assembled into scaffolds totaling ~71 Mb of data. B. malayi is estimated to have ~ 11,500 protein coding genes from ~ 71 Mb of assembled sequence and occupy ~32% of the sequence at an average density of 162 genes/ Mb (Ghedin et al., 2007) Brugia malayi microarray Complete sequencing of the genome of B. malayi was followed by the construction of oligonucletoide microarrays for expression profiling (Li et al., 2005; Li et al., 2006). With microarray technology, the expression levels of thousands of genes in a single experiment can be measured unlike with Northern blot analysis or PCR-based techniques (Gobert et al., 2005). Microarrays are specially produced slides that have thousands of individual DNA probes attached in an ordered array to the surface. The most commonly used microarrays are (1) 16

26 Complementary DNA (cdna) array: cdna probe is transferred to a glass slide by an arrayprinting machine and stored until use. (2) Oligonucleotide array: oligos are manufactured separately and then chips are facilitated by simple array printing machines. This makes this method inexpensive compared to others (Street, 2002). In both types, a known gene set is gridded onto a solid support medium in an ordered manner and probed with mrna/cdna from parasites exposed to different conditions of interest (Knox, 2004). Technological advances in robotics allow thousands of genes to be spotted onto glass microscopic slides. Nucleic acid probes can carry fluorescent labels (Cy3 and Cy5 dyes) which allow the direct analysis of differential gene expression with the aid of sophisticated image analysis software. Experiments can be repeated in a highly reproducible manner, which allows statistical validation of the output. New version 2 (V.2) microarray chips are available for B. malayi at Washington University School of Medicine, Microarray Core Facility. These chips are spotted with 18,153 oligos in total and each oligo is unique. The 18, 153 oligonucleotides on the version 2 (V.2) array include: 1. 15, 455 B. malayi oligos (accounts for 85% of total spots) 2. 1,016 O. volvulus oligos (6%; those not already represented above) oligos based on EST clustering of W. bancrofti data (5%; those not already represented above) endosymbiont bacteria Wolbachia oligos (4%; those not already represented above) (Dr. S.A. Williams, personal communication, 2008 and 17

27 1.7.3 Identifying genes in B. malayi that may be important in the development of resistance to albendazole To develop more effective strategies to monitor and prevent the development of resistance, a better understanding of the genetic and molecular basis of anthelmintic resistance in parasites is required. Gaining basic knowledge of gene expression in B. malayi and identifying differentially expressed genes in worms that are able to survive levels of ABZ that kill a majority of the worms exposed to the drug and comparing their gene expression levels may give a clearer picture about genes disregulated due to treatment with ABZ. It should also help to elucidate mechanisms involved in the action of ABZ on worms, and at the same time help determine, what allows some worms to survive doses of drugs that kill the majority of worms. Such knowledge may generate important insights into possible mechanisms of drug resistance. With the aid of microarray technology, identifying differentially expressed genes becomes feasible. Further characterization of those identified genes will be possible by other advanced molecular techniques like Real-time RT-PCR and RNA interference (RNAi). Having such powerful molecular tools to use on an important human nematode parasite that is so easily maintained in a rodent model provides an excellent and unique system for studying the biology of parasitic nematodes. This study will be an important step in gaining increased understanding of anthelmintic resistance at the molecular level. Furthermore, it is expected that findings will be applicable to other human filarial nematodes including W. bancrofti and O. volvulus and parasitic nematodes of livestock where anthelmintic resistance is now reaching critical and alarming levels. 18

28 References Addiss, D.G., Beach, M.J., Streit, T.G., Lutwick, S., LeConte, F.H., Lafontant, J.G., Hightower, A.W., Lammie, P.J., Randomised placebo-controlled comparison of ivermectin and albendazole alone and in combination for Wuchereria bancrofti microfilaraemia in Haitian children. Lancet (British edition) 350, Ah, H.S., Thompson, P.E., 1973a. Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus). Experimental Parasitology 34, Ah, H.S., Thompson, P.E., 1973b. Brugia pahangi: infections and their effect on the lymphatic system of Mongolian jirds (Meriones unguiculatus). Exp Parasitol 34, Albonico, M., Smith, P.G., Hall, A., Chwaya, H.M., Alawi, K.S., Savioli, L., A randomized controlled trial comparing mebendazole and albendazole against Ascaris, Trichuris and hookworm infections. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, Albonico, M., Bickle, Q., Ramsan, M., Montresor, A., Savioli, L., Taylor, M., Efficacy of mebendazole and levamisole alone or in combination against intestinal nematode infections after repeated targeted mebendazole treatment in Zanzibar. Bulletin of the World Health Organization 81, Albonico, M., Engels, D., Savioli, L., Monitoring drug efficacy and early detection of drug resistance in human soil-transmitted nematodes: a pressing public health agenda for helminth control. International Journal for Parasitology 34,

29 Anderson, T.J.C., Blouin, M.S., Beech, R.N., Population biology of parasitic nematodes: applications of genetic markers. Advances in Parasitology 41, Ash, L.R., Chronic Brugia pahangi and Brugia malayi infections in Meriones unguiculatus. J Parasitol 59, Babu, B.V., Nayak, A.N., Dhal, K., Acharya, A.S., Jangid, P.K., Mallick, G., The economic loss due to treatment costs and work loss to individuals with chronic lymphatic filariasis in rural communities of Orissa, India. Acta Trop 82, Bell, R.G., Adams, L., Coleman, S., Negrao-Correa, D., Klei, T., Brugia pahangi: quantitative analysis of infection in several inbred rat strains. Exp Parasitol 92, Besier, R.B., Love, S.C.J., Anthelmintic resistance in sheep nematodes in Australia: the need for new approaches. Australian Journal of Experimental Agriculture 43, Brown, H.D., Antiparasitic drugs. IV. 2-(4'thiazolyl)-benzimidazole, a new anthelmintic. Journal of the American Chemical Society 83, CDC, Recommendations of the International Task Force for Disease Eradication. In, Morb Mortal wkly Rep 42 (1993)(RR-16) Centers for Disease Control and Prevention pp Chirgwin, S.R., Rao, U.R., Coleman, S.U., Nowling, J.M., Klei, T.R., Profiling the cellular immune response to multiple Brugia pahangi infections in a susceptible host. Journal of Parasitology 91, Clercq, D.d., Sacko, M., Behnke, J., Gilbert, F., Dorny, P., Vercruysse, J., Failure of mebendazole in treatment of human hookworm infections in the southern region of Mali. American Journal of Tropical Medicine and Hygiene 57,

30 Crandall, R.B., McGreevy, P.B., Connor, D.H., Crandall, C.A., Neilson, J.T., McCall, J.W., The ferret (Mustela putorius furo) as an experimental host for Brugia malayi and Brugia pahangi. Am J Trop Med Hyg 31, Dasgupta, A., Cursory survey of lymphatic filariasis. An overview. Indian Journal of Pathology and Microbiology 27, Dhanda, V., Das, P.K., Lal, R., Srinivasan, R., Ramaiah, K.D., Spread of lymphatic filariasis, re-emergence of leishmaniasis and threat of babesiosis in India. Indian Journal of Medical Research 103, Dreyer, G., Addiss, D., Noroes, J., Amaral, F., Rocha, A., Coutinho, A., 1996a. Ultrasonographic assessment of the adulticidal efficacy of repeat high-dose ivermectin in bancroftian filariasis. Tropical Medicine and International Health 1, Dreyer, G., Santos, A., Norões, J., Rocha, A., Addiss, D., 1996b. Amicrofilaraemic carriers of adult Wuchereria bancrofti. Transactions of the Royal Society of Tropical Medicine and Hygiene 90, Driscoll, M., Dean, E., Reilly, E., Bergholz, E., Chalfie, M., Genetic and Molecular Analysis of a Caenorhabditis elegans β-tubulin That Conveys Benzimidazole Sensitivity. The Journal of Cell Biology 109, Drogemuller, M., Failing, K., Schnieder, T., von Samson-Himmelstjerna, G., 2004a. Effect of repeated benzimidazole treatments with increasing dosages on the phenotype of resistance and the beta-tubulin codon 200 genotype distribution in a benzimidazoleresistant cyathostomin population. Vet Parasitol 123,

31 Drogemuller, M., Schnieder, T., von Samson-Himmelstjerna, G., 2004b. Beta-tubulin complementary DNA sequence variations observed between cyathostomins from benzimidazole-susceptible and -resistant populations. J Parasitol 90, Eberhard, M.L., Hightower, A.W., Addiss, D.G., Lammie, P.J., Clearance of Wuchereria bancrofti antigen after treatment with diethylcarbamazine or ivermectin. American Journal of Tropical Medicine and Hygiene 57, Elard, L., Humbert, J.F., Importance of the mutation of amino acid 200 of the isotype 1 beta-tubulin gene in the benzimidazole resistance of the small-ruminant parasite Teladorsagia circumcincta. Parasitol Res 85, Eng, J.K.L., Prichard, R.K., A comparison of genetic polymorphism in populations of Onchocerca volvulus from untreated- and ivermectin-treated patients. Molecular and Biochemical Parasitology 142, Eng, J.K.L., Blackhall, W.J., Osei-Atweneboana, M.Y., Bourguinat, C., Galazzo, D., Beech, R.N., Unnasch, T.R., Awadzi, K., Lubega, G.W., Prichard, R.K., Ivermectin selection on β-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus. Molecular and Biochemical Parasitology 150, Evans, D.B., Gelband, H., Vlassoff, C., Social and economic factors and the control of lymphatic filariasis: a review. Acta Tropica 53, Freedman, D.O., Almeida Filho, P.J.d., Besh, S., Maia e Silva, M.C., Braga, C., Maciel, A., Furtado, A.F., 1995a. Abnormal lymphatic function in presymptomatic bancroftian filariasis. Journal of Infectious Diseases 171, Freedman, D.O., Bui, T., Almeida Filho, P.J.d., Braga, C., Maia e Silva, M.C., Maciel, A., Furtado, A.F., 1995b. Lymphoscintigraphic assessment of the effect of 22

32 diethylcarbamazine treatment on lymphatic damage in human bancroftian filariasis. American Journal of Tropical Medicine and Hygiene 52, Friedman, P.A., The molecular mechanism of action of benzimidazole drugs in embryos of Ascaris suum. Dissertation Abstracts International 40B, Geary, T.G., Nulf, S.C., Favreau, M.A., Tang, L., Prichard, R.K., Hatzenbuhler, N.T., Shea, M.H., Alexander, S.J., Klein, R.D., 1992a. Three beta-tubulin cdnas from the parasitic nematode Haemonchus contortus. Mol Biochem Parasitol 50, Geary, T.G., Nulf, S.C., Favreau, M.A., Tang, L., Prichard, R.K., Hatzenbuhler, N.T., Shea, M.H., Alexander, S.J., Klein, R.D., 1992b. Three β-tubulin cdnas from the parasitic nematode Haemonchus contortus. Molecular and Biochemical Parasitology 50, Ghedin, E., Wang, S., Foster, J.M., Slatko, B.E., 2004a. First sequenced genome of a parasitic nematode. Trends Parasitol 20, Ghedin, E., Wang, S.L., Foster, J.M., Slatko, B.E., 2004b. First sequenced genome of a parasitic nematode. Trends in Parasitology 20, Ghedin, E., Wang, S., Spiro, D., Caler, E., Zhao, Q., Crabtree, J., Allen, J.E., Delcher, A.L., Guiliano, D.B., Miranda-Saavedra, D., Angiuoli, S.V., Creasy, T., Amedeo, P., Haas, B., El-Sayed, N.M., Wortman, J.R., Feldblyum, T., Tallon, L., Schatz, M., Shumway, M., Koo, H., Salzberg, S.L., Schobel, S., Pertea, M., Pop, M., White, O., Barton, G.J., Carlow, C.K., Crawford, M.J., Daub, J., Dimmic, M.W., Estes, C.F., Foster, J.M., Ganatra, M., Gregory, W.F., Johnson, N.M., Jin, J., Komuniecki, R., Korf, I., Kumar, S., Laney, S., Li, B.W., Li, W., Lindblom, T.H., Lustigman, S., Ma, D., Maina, C.V., Martin, D.M., McCarter, J.P., McReynolds, L., Mitreva, M., Nutman, T.B., Parkinson, J., Peregrin-Alvarez, J.M., Poole, C., Ren, Q., Saunders, L., Sluder, A.E., Smith, K., Stanke, 23

33 M., Unnasch, T.R., Ware, J., Wei, A.D., Weil, G., Williams, D.J., Zhang, Y., Williams, S.A., Fraser-Liggett, C., Slatko, B., Blaxter, M.L., Scott, A.L., Draft genome of the filarial nematode parasite Brugia malayi. Science 317, Gigliolo, G., Filariasis in British Guiana. Some industrial medical problems.. Indian Journal of Malariology 14, Gilleard, J.S., Understanding anthelmintic resistance: The need for genomics and genetics. International Journal for Parasitology 36, Gilleard, J.S., Beech, R.N., Population genetics of anthelmintic resistance in parasitic nematodes. Parasitology 134, Gobert, G.N., Moertel, L.P., McManus, D.P., Microarrays: new tools to unravel parasite transcriptomes. Parasitology 131, Grant, W.N., Mascord, L.J., Beta-tubulin gene polymorphism and benzimidazole resistance in Trichostrongylus colubriformis. International Journal for Parasitology 26, Guenette, S., Prichard, R.K., Klein, R.D., Matlashewski, G., Characterization of a betatubulin gene and a beta-tubulin gene products of Brugia pahangi. Mol Biochem Parasitol 44, Haddix, A.C., Kestler, A., Lymphatic filariasis: economic aspects of the disease and programmes for its elimination. Trans R Soc Trop Med Hyg 94, Herlich, H., Rew, R.S., Colglazier, M.L., INHERITANCE OF CAMBENDAZOLE RESISTANCE IN HAEMONCHUS-CONTORTUS. American Journal of Veterinary Research 42,

34 Hunter, J.M., Elephantiasis: a disease of development in north east Ghana. Social Science and Medicine 35, Ismail, M.M., Jayakody, R.L., Weil, G.J., Nirmalan, N., Jayasinghe, K.S.A., Abeyewickreme, W., Sheriff, M.H.R., Rajaratnam, H.N., Amarasekera, N., Silva, D.C.L.d., Michalski, M.L., Dissanaike, A.S., Efficacy of single dose combinations of albendazole, ivermectin and diethylcarbamazine for the treatment of bancroftian filariasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 92, Kaplan, R.M., Drug resistance in nematodes of veterinary importance: a status report. Trends in Parasitology 20, Kessel, J.F., Disabling effects and control of filariasis. Am J Trop Med Hyg 6, ; discussion, 415. Knox, D.P., Technological advances and genomics in metazoan parasites. International Journal for Parasitology 34, Kwa, M.S.G., Kooyman, F.N.J., Boersema, J.H., Roos, M.H., 1993a. EFFECT OF SELECTION FOR BENZIMIDAZOLE RESISTANCE IN HAEMONCHUS-CONTORTUS ON BETA- TUBULIN ISOTYPE-1 AND ISOTYPE-2 GENES. Biochemical and Biophysical Research Communications 191, Kwa, M.S.G., Veenstra, J.G., Roos, M.H., 1993b. Molecular characterisation of β-tubulin genes present in benzimidazole-resistant populations of Haemonchus contortus. Molecular and Biochemical Parasitology 60, Kwa, M.S.G., Veenstra, J.G., Roos, M.H., BENZIMIDAZOLE RESISTANCE IN HAEMONCHUS-CONTORTUS IS CORRELATED WITH A CONSERVED 25

35 MUTATION AT AMINO-ACID-200 IN BETA-TUBULIN ISOTYPE-1. Molecular and Biochemical Parasitology 63, Kwa, M.S.G., Veenstra, J.G., Vandijk, M., Roos, M.H., BETA-TUBULIN GENES FROM THE PARASITIC NEMATODE HAEMONCHUS-CONTORTUS MODULATE DRUG- RESISTANCE IN CAENORHABDITIS-ELEGANS. Journal of Molecular Biology 246, Lacey, E., The role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. Int J Parasitol 18, Lacey, E., Mode of action of benzimidazoles. Parasitology Today 6, Lacey, E., Gill, J.H., 1994a. Biochemistry of benzimidazole resistance. Acta Tropica 56, Lacey, E., Gill, J.H., 1994b. Biochemistry of benzimidazole resistance. Acta Trop 56, Lawrence, R.A., Lymphatic filariasis: what mice can tell us. Parasitol Today 12, Lejambre, L.F., Royal, W.M., Martin, P.J., INHERITANCE OF THIABENDAZOLE RESISTANCE IN HAEMONCHUS-CONTORTUS. Parasitology 78, Li, B.W., Rush, A.C., Crosby, S.D., Warren, W.C., Williams, S.A., Mitreva, M., Weil, G.J., Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cdna oligonucleotide array analysis. Molecular and Biochemical Parasitology 143, Li, B.W., Rush, A.C., Weil, G.J., McCarter, J.P., Mitreva, M., Brugia malayi: effects of radiation and culture on gene expression in infective larvae. Molecular and Biochemical Parasitology 149,